The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FA-competent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors. © 2008 Wiley-Liss, Inc.